The stimulatory (Gs) and inhibitory (Gi) G proteins regulate the activity of adenylyl cyclase (AC). Activation of these heterotrimeric (alpha beta gamma) G proteins occurs when an agonist-receptor complex promotes the exchange of GDP for GTP in the nucleotide binding site of the alpha- subunit (Galpha). Under some circumstances, Galpha dissociates from the beta gamma-subunit complex (Gbeta gamma). The prevailing hypothesis proposes that subunit dissociation necessarily accompanies G protein activation, and subunit reassociation accelerates G protein deactivation by stimulating the intrinsic GTPase activity of Galpha. Thus, the interplay between Gsalpha , Gialpha and Gbeta gamma is critical for controlling the activity of AC. However, subunit dissociation may not accompany Gs activation. Gs was prepared from bovine brain and its activity determined by reconstituting AC in Gsalpha-deficient cyc- membranes. Gs subunit dissociation was assayed by immunoprecipitating Gsalpha, and determining the amount of Gbeta that was coprecipitated, or by the ability of Gs to serve as a substrate for choleragen (CT). CT ADP-ribosylates heterotrimeric Gs but not the free Gsalpha subunit. We found that in solution with 2 mM Mg2+, both GTPgammaS and AIF4- activated Gs without causing subunit dissociation. In solution, Mg2+ (2-120 mM) caused a dose-dependent dissociation of Gs subunits that was inhibited to the same extent by GDP and GTP. 120 mM Mg2+ caused nearly complete Gs subunit dissociation, and inactivated Gs unless guanine nucleotides were present. Gs was incubated in solution with 120 mM Mg2+ for 0-2 h in the absence of guanine nucleotides, then GTPgammaS was added to stop further inactivation, and to activate any Gs that was not denatured. Gs activated in this way could not be ADP-ribosylated by CT in solution indicating that complete subunit dissociation had occurred(Gs alpha - GTPgammaS plus Gbeta gamma). When Gs alpha-GTPgammaS was incorporated into cyc-, it became a substrate for CT and also stimulated AC. These two phenomenon were closely correlated and inversely related to the length of exposure to 120 mM Mg2+ in the absence of GTPgammaS. In vitro transcription and translation was used to prepare active Gs alpha (Gs alpha52) and type IV adenylyl cyclase (ACIV) from recombinant DNA. In solution, Gs from bovine brain could activate ACIV, but activation by Gs alpha52 was largely dependent upon Gbeta gamma. Gbeta gamma alone had no effect on the activity of ACIV. When cyc- were depleted of Gbeta gamma by a detergent/salt wash, and reconstituted with Gs alpha52, it was a poor activator of AC and a poor substrate for CT. If Gbeta gamma was included during reconstitution, it caused a concentration-dependent increase in both AC activity and ADP-ribosylation of Gs alpha52, and these two phenomenon were closely correlated. These data suggest that the activated Gs heterotrimer, and not the free Gs alpha subunit mediates stimulation of AC both in membranes and in solution.